La kinase dépendante des cyclines 5 (Cdk5) est un facteur de sensibilité aux inhibiteurs de PARP et aux rayonnements ionisants (RI), elle est nécessaire pour le point de contrôle du cycle cellulaire en phase S. Cependant, elle n’est pas directement impliquée dans la réparation des cassures de brin d’ADN, suggérant un rôle dans les étapes plus précoces de la signalisation des dommages. Nous rapportons ici que des cellules HeLa déplétées pour Cdk5 (Cdk5 KD) montrent une grande sensibilité aux RI surtout lorsqu'elles sont irradiées en phase S, au 5-Fluoro-Uracile, à la 6-Thioguanine et à une exposition chronique à l'hydroxyurée (HU). Les cellules Cdk5 KD montrent une altération de la dynamique de la phase S causée par une vitesse de réplication plus lente et une réduction des origines actives par mégabase d'ADN. En revanche, après un traitement au HU, ces cellules sortent plus rapidement du blocage en phase S. Ceci s’accompagne d’une déficience de la phosphorylation de RPA-32 sur les sérines 29 et 33 et de SMC1 sur la sérine 966 ainsi que d’une réduction du niveau de dommages de l'ADN évalués par le test des comètes alcalines, de l’intensité du signal gamma-H2AX, des foyers RPA, Rad51 et RPA sur les sérines 4 et 8 ainsi que du niveau d'échanges de chromatides sœurs. Des essais kinase in vitro couplés à la spectrométrie de masse ont montré que Cdk5 peut phosphoryler RPA-32 sur ses sérines 23, 29, et 33. De plus, des niveaux inférieurs d’expression de Cdk5 ont été associés à une meilleure survie sans métastases chez des patientes atteintes d’un cancer du sein et à une réduction de la survie des cellules de tumeurs du sein déplétées pour Cdk5 après un traitement aux RI et en présence d’un inhibiteur de PARP. Globalement, ces résultats montrent que Cdk5 est nécessaire pour la réplication basale et l'activation du point de contrôle en phase S en réponse à un stress réplicatif, ouvrant des perspectives cliniques intéressantes pour améliorer la destruction des cellules tumorales dans certaines populations de patientes atteintes de cancer du sein grâce à des agents qui génèrent un stress réplicatif. / Cyclin dependent kinase 5 (Cdk5) is a determinant of sensitivity to PARP inhibitors and ionizing radiation (IR) and is required for the intra-S DNA damage checkpoint. It is not however directly implicated in strand break repair suggesting a role in the earlier steps of checkpoint activation. We report here that Cdk5-Depleted (Cdk5-KD) HeLa cells show higher sensitivity to IR when irradiated in S-Phase, and to chronic hydroxyurea (HU) exposure, 5-Fluorouracil and 6-Thioguanine. Cdk5-KD cells show altered basal S-Phase dynamics caused by a slower replication velocity and fewer active origins per megabase of DNA, however they show a faster recovery from an HU block. This was accompanied by impaired RPA-32 priming serine 29 and serine 33 phosphorylations and SMC1-Serine 966 phosphorylation as well as lower levels of DNA damage assessed by the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci and RPA-32 serine 4 and serine 8 phosphorylation and levels of sister chromatid exchanges. In vitro kinase assays coupled with mass spectrometry showed that Cdk5 can phosphorylate RPA-32 on serines 23, 29, and 33. In addition lower Cdk5 levels were associated with longer metastasis free survival in breast cancer patients and lower cell survival in Cdk5 depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and opens up interesting clinical opportunities to enhance tumor cell killing in certain populations of breast cancer patients through agents that generate replication stress.

Orientador: Frab Norberto Boscolo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-07-22T03:00:44Z (GMT). No. of bitstreams: 1 Almeida_SolangeMariade_D.pdf: 5623908 bytes, checksum: b26c68566d3a5cc890cf18e225a7c0c1 (MD5) Previous issue date: 1997 / Resumo: O presente trabalho teve como finalidade estudar o efeito de baixas doses de radiação de elétrons no processo de reparação tecidual em ratos. Para tanto, os animais sofreram um procedimento cirúrgico, onde foi produzida uma ferida retangular, medindo 2,3 cm por 1,4 cm, na sua região dorsal anterior. No momento da irradiação, as feridas produzidas foram protegidas, sendo irradiada somente uma região corresponde a 1,0 cm lateralmente à cada borda da ferida, com todo o restante do corpo do animal também protegido. A irradiação foi realizada para um grupo de animais, imediatamente após a abertura da ferida. O outro grupo sofreu a irradiação 3 dias após esse procedimento. O processo de reparação tecidual foi estudado aos 2, 4, 7, 11, 14, 17 e 21 dias após o procedimento cirúrgico para o primeiro grupo, enquanto para o segundo grupo de animais, a reparação tecidual foi avaliada 5, 7, 10, 14, 17, 20 e 24 dias também após a abertura da ferida. Cada grupo irradiado foi comparado com. grupos controles correspondentes, os quais não sofreram irradiação. O processo de reparação tecidual foi avaliado pelos seguintes métodos: coloração pela hematoxilina - eosina, que possibilitou avaliar a mortologia do tecido de granulação; reação histoquímica de metacromasia pelo azul de toluidina pH 4, podendo assim ser avaliada a síntese de glicosaminoglicanas e por fim, impregnação argêntica, onde foi observada a síntese de colágeno, através da microscopia de polarização (birrefringência). Os resultados obtidos mostraram que 1,0 Gy de radiação de elétrons com um feixe de 6 MéV, usou um retardo no processo de reparação tecidual, quando aplicado imediatamente e 3 dias após a abertura da ferida, sendo que quando comparados os dois grupos irradiados, para os dias 7, 14 e 17 , o efeito na reparação tecidual foi mais acentuado no grupo que sofreu irradiação 3 dias após a abertura da ferida / Abstract: The present search had the purpose to study the low dose electron irrradiation effect in the process of tissue repair in rats. In such a way, the animais were submitted to a surgical procedure, in which a rectangular wound was performed, measuring 2.3cm X 1.4cm on the fore dorsal area. At the moment of irradiation, the wounds were protected so that only an area near 1.0cm laterally to each b9rder of the wound was i rrad iated , being protected ali the rest ofthe animal body. The irradiation was performed in one group of animais immediately after the wounding procedure. The other group was irradiated three days after wounding. The process of tissue repair was studied at 2, 4, 7, 11, 14, 17 and 21 days after the surgical procedure on the first group, while for the other group of animais, tissue repair was evaluated at 5, 7, 10, 14, 17, 20 and 24 days, also after wounding. Each irradiated group was compared to corresponding control groups, which did not were submited irradiation. The tissue repair process was evaluated by the following methods: staining by haematoxylin-eosin in order to evaluating granulation tissue morphology; histochemical reaction of metachromasia by toluidin pH 4.0, so that it was possible to evaluate the synthesis of glucosaminglucans and at last, the silver impregnation, in which it was studied the collagen synthesis bymeans of polarizing microscopy. The results obtained showed that 1.0 Gy of electron irradiation with a 6 MeV beam caused a delay in the process of tissue repair, when applied immediately after and at three days after wounding. The comparison of both irradiated groups at days 7, 14 and 17, have showed that the effect on tissue repair was stronger on the group that received irradiation 3 days after wounding / Doutorado / Radiologia / Doutor em Odontologia